Techniques for specifically amplifying a target nucleic acid sequence are very important for research and clinical applications (e.g. genetic testing) in molecular biology. An amplification product obtained by such a nucleic acid amplification technique can be specifically detected, for example, by a detection method using a target sequence-containing nucleic acid fragment immobilized on a solid phase. This method, which is designed to specifically capture a target nucleic acid on the solid phase, allows for easy removal of non-specific nucleic acids by washing or the like, and thus improves the detection specificity.
In this method, in order to capture a target nucleic acid on the solid phase, a technique using an antigen-antibody or ligand-receptor pair capable of specifically binding together may be used. For example, Non Patent Literature 1 discloses a method for detecting a product of PCR amplification using a primer terminally modified with biotin and another primer modified with a fluorescent substance. In this method, the PCR product is contacted with a solid phase containing streptavidin and agarose, and then bonded to the solid phase as a streptavidin-biotin complex, which can be measured for fluorescence to assay the target amplification product.
Unfortunately, the number of antigen-antibody or ligand-receptor combinations usable for labeling is limited, which makes it substantially difficult to simultaneously detect multiple target nucleic acids. Another problem is the cost: fluorescently labeled nucleic acids are expensive.
Another technique for capturing a target nucleic acid on a solid phase is to immobilize a probe containing an oligonucleotide having a complementary sequence to the target nucleic acid on the solid phase, which enables the target nucleic acid to be indirectly immobilized on the solid phase through hybridization of the target nucleic acid and the probe. This technique is accompanied with detection of the intensity of a signal of the formed hybrid. This type of nucleic acid analysis makes it possible to simultaneously assay multiple target sequences by using varied probe sequences.
In general, however, hybridization of an immobilized probe and a target nucleic acid on a solid phase requires heating treatment for denaturing a double-stranded nucleic acid amplified by PCR into single strands. Unfortunately, this heating treatment is troublesome and is also associated with a reduction in hybridization efficiency due to reannealing. Another problem is that single-stranded DNA tends to curl into a ball and is thus inferior in detection sensitivity. Although Patent Literature 1 discloses a technique for amplifying a single-stranded nucleic acid via nuclease treatment without heating treatment, this technique is also a troublesome procedure and has the problem of curling of single strands into balls.
Among nucleic acid detection methods, the method based on chromatography disclosed in Patent Literature 2 is easy to operate and allows for rapid and easy detection of target nucleic acids. This is a gene detection method that includes the steps of sampling genes from a cell, virus or bacterium, fragmenting the randomly sampled genes, and detecting a target gene, wherein these steps are continuously performed on a single device for gene detection by transferring a liquid sample containing the randomly sampled genes or fragments thereof by capillary action. This method allows not only assessment of the presence of a target gene but also identification of its type.
Also in Patent Literature 2, however, single-stranded nucleic acids are amplified by NASBA. The problems in the use of single-stranded nucleic acids are as described above.
In order to solve the above problems, Patent Literatures 3 and 4 disclose that a non-natural nucleic acid tag, a hairpin structure or a pseudoknot structure for inhibiting nucleic acid synthesis by DNA polymerase is present on the 5′ side of the primer region. Thus the single-stranded region is left at one end of the double-stranded nucleic acid after PCR reactions. This technique is advantageous in that an amplified double-stranded DNA product having a hybridizable single-stranded region at one end of the double-stranded DNA can be produced only by performing PCR reactions using such a special primer. However, since a fluorescent label or surface plasmon resonance (SPR) imaging is required for detection, expensive special equipment is necessary. Additionally, there are problems with speed and simplicity.